Chapter -3 Materials
& Methods
TTTThe present investigation has been undertaken to evaluate the role of
Tribulus terrestris root and fruit extract in modifying the lead acetate induced
toxicity in the liver and blood of Swiss albino mice.
The materials and methodology employed in the present investigation are as
under-
1. Animals selection
Swiss albino mice (Mus musculus) procured from IVRI - Izatnagar, India,
were bred in an animal house under controlled conditions of temperature (25±3oC)
and light (12 hrs. light and 12 hrs. darkness). These animals were given pelleted
standard mice feed (obtained from Hindustan Lever Ltd., Delhi) and water ad
libitum. A total check on cleanliness of the cages and animals were observed
throughout the experimental period. Occasionally soaked black gram and wheat
were given as supplementary diet. Tetracycline water once a fortnight was given to
these animals as one of the preventive measures against any infection.
A close-bred colony of these animals has been maintained by random
breeding. For this purpose, adult female and male mice were kept in the ratio of 3:1
polypropylene cages (15" × 10" × 6 size) having a wire gauge top. Sawdust was
used as breeding material in the cages.
Healthy young mice 6-8 weeks old with an average body weight (22±2 gm)
selected from the above colony were used for the present study.
2. Source of heavy metal toxicant
Heavy metal lead in the form of lead acetate was used for the present study,
it was obtained from Central Drug House (India). Lead acetate was dissolved in
vehicle Double Distilled Water (DDW) and administered subcutaneously.
3. Source of plant extract
The plant Tribulus terrestris (T. terrestris) was collected locally in the month
of July and August from the University of Rajasthan campus and was identified in
the Herbarium of Botany Department, University of Rajasthan, Jaipur as an RUBL-
Tribulus terrestris (RUBL 20824)
fruit
root
Plate A
Material and Methods 33
20824 variety (Plate A). The roots and fruits of the plant were air dried in shade and
powdered. Both the powders were distilled separately in soxhlet apparatus (for 36
hours using DDW) at 40°C. The remaining material was dried in oven at 36°C and
was used for the study. The dose was administrated through oral gavage to the mice.
4. Evaluation of antioxidant variables from Tribulus terrestris root and
fruit extract
DPPH radical scavenging assay
DPPH radical scavenging activity was determined by the method of Blois
(1958).
Principle: DPPH is a stable radical of purple color which is reduced to
yellow colored diphenylpicrylhydrazine, when it reacts with an antioxidant
compound which can donate hydrogen. The free radical scavenging activity of T.
terrestris root and fruit extract was measured in terms of hydrogen donating or
radical scavenging ability using the stable radical DPPH.
Procedure
i. 0.1 mM solution of DPPH in ethanol was prepared and 1.0 ml of this
solution was added to 3.0 ml of extract solution in water at different
concentrations (10-60 µg/ml).
ii. Thirty minutes later the absorbance was measured at 517 nm.
Lower absorbance of the reaction mixture indicates higher free radical
scavenging activity. Ascorbic acid was used as the standard antioxidant compound.
The capability to scavenge the DPPH radical was calculated using the following
equation:
DPPH Scavenged (%) = Acont - Atest x 100
Acont Where Acont is the absorbance in the presence of the control reaction and Atest is the
absorbance in the presence of the sample of the extracts.
Material and Methods 34
5. Drug tolerance study & optimum dose selection of T. terrestris root and
fruit extract
Tribulus terrestris root and fruit extracts at the dose level of 800 mg/kg b.wt.
were given orally for 30 days. This dose was selected on the basis of previous
studies done in our lab. Mice for drug tolerance study were divided into various
groups of 5 animals each, out of which one is control and remaining are treatment
groups. Treatment groups were given 100, 400 and 800 mg/kg body weight/day T.
terrestris root and fruit extracts in double distilled water by oral gavage for 7
consecutive days. All these animals were observed regularly till 30 days for any sign
of morbidity, mortality and behavioural toxicity. Maximum tolerance doses of T.
terrestris root and T. terrestris fruit extracts were determined accordingly by
evaluating LPO (Lipid peroxidation) level and GSH (reduced glutathione) level in
mouse liver.
6. Dose selection of lead acetate
Lead was given subcutaneously in the form of lead acetate at different dose
levels (0, 5, 10, 20 and 40 mg/kg body weight) only once.
The dose was decided on the basis of body weight changes, and GSH and
LPO levels in the liver. After administration of 5 mg/kg b.wt. lead acetate
subcutaneously in Swiss albino mice, no significant changes were found in body
weight, GSH and LPO levels of liver tissue and the values were very similar to the
normal control values after 7 days. On the dose level of 10 mg/kg body weight,
slight changes were observed with decreased body weight and GSH level and
increased LPO level as compared to normal control. Symptoms of sickness, ruffled
hair and loss of appetite were also observed after 7 days but no mortality was
observed up to 30 days of experiment. While on dose levels 20 and 40 mg/kg b.wt.,
body weight and GSH level was decreased more and LPO level was increased
highly as compared to normal control. Symptoms of sickness were more severe and
localized infection of skin (skin lesions) after lead-intoxication subcutaneously was
also observed on these dose levels but no skin lesions were observed on 5 and 10
Normal Swiss albino mice
Ruffled hair Swiss albino mice
Swiss albino mice with skin lesion
Plate B
Material and Methods 35
mg/kg b.wt. doses (Plate B). On the basis of above study for dose determination of
lead acetate, we selected a dose of 10 mg/kg body weight for our experiment.
Table A: Showing body weight changes of animals during 7 days of experiment
Groups Lead acetate (mg/kg b. wt.)
Body weight after lead acetate treatment (gm)
1st day 3rd day 5th day 7th day
I Normal Control (DDW orally)
30±1.80 30±1.20 30.5±1.60 30.5±0.90
II 5 mg/kg 30.5±0.90 30±1.40 30±1.20 29.5±1.80
III 10 mg/kg 31±2.40 30±1.70 28.5±0.70 27±1.90
IV 20 mg/kg 30±1.30 28.5±2.10 27±1.80 25±1.60
V 40 mg/kg 29.5±1.70 27±1.40 25.5±0.90 24±1.20
Table B: Showing GSH and LPO changes on different lead acetate doses in
liver of animals during 7 day experiment
Groups Lead acetate (mg/kg b. wt.)
No. of Animals
GSH level in liver (mg GSH/gm
tissue)
LPO level in liver (µMTBARS/mg
protein)
I Normal Control (DDW orally)
5 8.03±0.41 3.79±0.15
II 5 mg/kg 5 7.59±0.11 4.11±0.10
III 10 mg/kg 5 6.67±0.23 4.88±0.13
IV 20 mg/kg 5 6.51±0.31 5.61±0.23
V 40 mg/kg 5 5.99±0.33 6.63±0.11
7. Normal Studies
The animals did not receive lead acetate or root and fruit extract (Tribulus
terrestris). They were autopsied to study the normal structure of liver.
Experimental Protocol
Group I (Control group): Normal control, untreated (mice received DDW as
vehicle).
Group IIa and IIb ( T. terrestris group): Selected dose of T. terrestris root (IIa) and
fruit extract (IIb) dissolved in DDW was administered orally upto 30 days.
Material and Methods 36
Group III (Heavy metal treated group): Freshly dissolved lead acetate in 0.1 ml
double distilled water was given subcutaneously only once at a dose of 10mg/kg
body weight. This day was considered as day zero and the experiment was continued
for 30 days.
Group IVa and IVb ( T. terrestris + Lead acetate + T. terrestris): T. terrestris root
and fruit extracts were given at a selected dose level (800mg/kg body weight) for 7
days and on the 7th day just after 30 minutes of T. terrestris root and fruit extract
administration lead acetate was given only once, this day was considered as day
zero. Then from the next day T. terrestris was continued for 30 days. The total
experimental period was of 37 days.
Group V [Vitamin C + Lead acetate + Vitamin C (positive control group)]:
Vitamin C was administered at a dose level of 100 mg/kg body weight for 7 days
and on the 7th day just after 30 minutes of Vitamin C administration lead acetate
was given only once, this day was considered as day zero. Then from the next day
Vitamin C was continued for 30 days. The total experimental period was of 37 days.
Group VI [Lead Acetate + DMSA (for 7 days)]: DMSA was administered at a
dose level of 50 mg/kg body weight for 7 days after 30 minutes of lead acetate
treatment.
Group VIIa and VIIb ( T. terrestris + Lead acetate + DMSA + T. terrestris
group): T. terrestris was administered orally for 7 days and on the7th day just after
30 minutes of T. terrestris root and fruit extract administration lead acetate was
given only once, this day was considered as day zero. Then from the next day
DMSA was given for 7 days only and T. terrestris was continued for 30 days. The
total experimental period was of 37 days.
Autopsy Intervals
At least 5 animals from all the above groups were sacrificed under light
chloroform anesthesia on 1, 3, 7, 15 and 30 days. Liver, blood and serum were taken
and processed for various histopathological and biochemical parameters.
Material and Methods 37
Table C: Design of Experiment
Groups Experimental Groups
No. of Animals
Dose Days of Treatment
I Normal control 25 DDW (orally) Upto 30 days
IIa T. terrestris (root) treated
25 T. terrestris root extract (800mg/kg b.
wt.)
Upto 30 days
IIb T. terrestris (fruit) treated
25 T. terrestris fruit extract (800mg/kg b.
wt.)
Upto 30 days
III Lead acetate treated 25 10 mg/kg b.wt. (in DDW, subcutaneously
only once)
30 day experiment
IVa T. terrestris + Lead acetate + T. terrestris
(root extract)
25 800 mg/kg b.wt. T. terrestris + 10 mg/kg
b. wt. lead acetate + T. terrestris
7 days T. terrestris + lead acetate on
7th day + T. terrestris for 30
days
IVb T. terrestris + Lead acetate + T. terrestris
(fruit extract)
25 800 mg/kg b.wt. T. terrestris+ 10 mg/kg
b. wt. lead acetate + T. terrestris
7days T. terrestris +lead acetate on
7th day+ T. terrestris for 30
days
V Vitamin C+ Lead acetate + Vitamin C
25 100 mg/kg b.wt. Vitamin C+ 10 mg/kg b. wt. lead acetate +
Vitamin C
7days Vitamin C + lead acetate on 7th day + Vitamin C
for 30 days
VI Lead acetate + DMSA
25 10 mg/kg b. wt. lead acetate + 50 mg/kg b.
wt. DMSA
1st day Lead acetate + up to 7 days DMSA (30 day experiment)
VIIa T. terrestris +Lead acetate+ DMSA and
T. terrestris(root extract)
25 800 mg/kg b.wt. T. terrestris + 10 mg/kg b. wt. lead acetate +
DMSA &T. terrestris
7days T. terrestris + lead acetate on 7th day + DMSA for 7 days and T. terrestris for 30
days
VIIb T. terrestris +Lead acetate+ DMSA and
T. terrestris (fruit extract)
25 800 mg/kg b.wt. T. terrestris+ 10 mg/kg b. wt. lead acetate +
DMSA &T. terrestris
7days T. terrestris + lead acetate on 7th day + DMSA for 7 days and T. terrestris for 30
days
Material and Methods 38
Parameters studied
To study the toxic effects of lead and the modulatory effects of T. terrestris
against lead induced alterations, following parameters were assessed:
A. General Parameters
1. Mortality
2. Sickness
3. Body weight
4. Liver weight
B. General Toxicity:
To assess the toxicity different histopathological, biochemical and
haematological parameters were done.
I. Histological Studies (Normal and Histopatholgoical)
Liver from autopsied animals were excised out and fixed in Bouin's fixative
for 24 hrs. The fixed tissue were further processed by standard method and sections
were cut at 5µ and stained with Haematoxylin and Eosine.
II. Biochemical Parameters
For all biochemical estimation, liver was taken out from animals and
homogenates were prepared. The parameters selected for biochemical study were as
follows -
i. Lipid Peroxidation (LPO) (Ohkawa et al.,1979)
ii. Reduced Glutathione (GSH) (Moron et al.,1979)
iii. Total Protein (Lowry et al., 1951)
iv. Glycogen (Montgomery, 1957)
v. Superoxide Dismutase (SOD) (Marklund and Marklund, 1974)
vi. Catalase (CAT) (Aebi, 1984)
vii. Glutathione Peroxidase (GPx) (Paglia and Valentine, 1967)
viii. Glutathione-S-Transferase (GST) (Habig et al., 1974)
Material and Methods 39
Liver Function Tests
i. Serum Alkaline Phosphatase (Kind and King, 1954)
ii. Serum Glutamate Oxaloacetate Transaminase (Reitman and Frankel, 1957)
iii. Serum Glutamate Pyruvate Transaminase (Reitman and Frankel, 1957)
Haematological Study
i. Haemoglobin concentration (Wintrobe et al., 1981)
Homogenate Preparation for GSH and LPO:
1 gm of tissue and 9 ml of Tris KCl were mixed with the help of pestle
mortar and sea sand. 10% homogenate w/v of tissue in Tris KCl was prepared.
i. Lipid Peroxidation (LPO) assay
Lipid peroxidation level in liver was estimated by the method of Ohkawa et
al. (1979) as thiobarbituric acid reactive substances. The concentration of TBARS
was expressed as n moles of malondialdehyde per mg of tissue using 1, 1, 3, 3 -
tetromethoxy propane (TMP) as the standard.
Principle : Malondialdehyde formed from the breakdown of polyunsaturated fatty
acids serves as a convenient index for determining the extent of the peroxidation
reaction. Peroxidation of lipids generates MDA, which reacts with thiobarbituric
acid gives a red species absorbing 532 nm.
Procedure:
i. 0.8 ml of tissue sample was taken in test tubes and to the 0.2 ml of 8.1% of
SDS, 1.5 ml of 20% acetic acid was added and the pH was adjusted to 3.5
with NaOH.
ii. To the above solution 1.5 ml of 0.6% aqueous solution of TBA was added.
iii. The mixture was made upto 4ml with distilled water (0.6 ml) and then heated
at 95°C for 60 min.
iv. After cooling in tap water, 1 ml of distilled water and 5 ml of n-butanol and
pyridine (15% µ/v) were added and shaken vigorously.
Material and Methods 40
v. The solution was centrifuged at 4000 rpm for 10 minutes. The organic layer
was taken and its absorbance was measured at 532 nm using UV-VIS
systronic spectrophotometer.
Standard Curve: A standard curve was prepared by using tetromethoxy-propane
(TMP, mol. wt. = 164.20) purchased from Lancaster, England). 1 to 12 n mol of
TMP was taken in corresponding no. of test tubes with all the reagents (minus
sample) were added. Standard curve was plotted by taking the absorbance against
TMP concentration on the abscissa.
ii. Reduced Glutathione (GSH) assay
The GSH level in liver was determined by the method of Moron et al.
(1979).
Principle: The reduced glutathione reacts with DTNB and forms a yellow coloured
complex with DTNB that absorbs at 412 nm.
Reduced glutathione + DTNB → 5-thio-2-nitrobenzoate (yellow)
The tissue homogenates (10% w/v) were immediately precipitated with 0.1
ml of 25% trichloroacetic acid (TCA), and the precipitate was removed after
centrifugation.
Free endogenous -SH was assayed in a total 3 ml volume by the addition of 2
ml of 0.5m M DTNB prepared in 0.2 M phosphate buffer (pH = 8.0) to 0.1 ml of the
supernatant, and absorbance was read at 412 nm using a UV-VIS systronic
spectrophotometer. GSH was used as an standard to calculate µ mole GSH /gm
tissue.
Homogenate Preparation for Total Protein, SOD, CAT, GPx and GST:
1 mg (or 0.001 gm) of tissue and 1 ml of double distilled water were mixed
with the help of pestle mortar and sea sand.
iii. Total Protein assay
Total protein content in liver was estimated by Lowry et al.(1951) method.
Material and Methods 41
Principle: Here, protein is precipitated with trichloroacetic acid. When Folin
Ciocatteu reagent (Phosphatungstic phosphomolybdic acid) is added, a complex
results, the intensity of which accounts for the amount of proteins present in tissue
Folin Ciocatteu reagent after 50 percent dilution in double distilled water.
Procedure
i. 0.5 ml of 10% TCA was added was added to 0.5 ml of homogenate
ii. It was centrifuged for 15 minutes and the supernatant was discarded.
iii. Precipitate was then washed with 1 ml of 5% TCA.
iv. It was again centrifuged and the supernatant was discarded.
v. Precipitate was washed twice with 2 ml of GDW each time.
vi. It was further centrifuged and the supernatant discarded.
vii. Precipitate was dissolved in 4 ml of 1 N NaOH.
viii. Test tubes were placed in a water bath/oven at 60-70° for 10 minutes.
ix. After cooling and centrifuging, 0.5 ml of the above liquid was added to 4.5
ml of Lowry's solution.
x. Finally 0.5 ml of 1 N Folin-Phenol reagent was added and mixed well.
xi. Reading was taken at 640 nm against blank solution.
Blank solution: 0.5 ml of 1 N NaOH + 4.5 ml of Lowry's solution + 0.5 ml of
Folin-Phenol reagent.
Calculation: Amount of Total Protein was calculated by the following formula:
Reading of unknown ×
Concentration of standard × 1000 ×
4
Reading of standard Tissue taken 0.05
Where:
4/0.05 is volume taken.
Protein content was expressed as mg of protein/gm of tissue.
iv. Glycogen assay:
The Glycogen content in liver was estimated by Montgomery(1957)
Material and Methods 42
Procedure:
i. 0.1 gm tissue was digested in 2 ml of 30% KOH and kept in boiling water
for 2-3 minutes.
ii. The digested tissue was brought to room temperature by cooling and 2.4 ml
absolute alcohol was added and left for 10 minutes.
iii. It was centrifuged was 30 minutes and supernatant was discarded and again
2.4 ml absolute alcohol was added.
iv. It was again centrifuged for 10-15 minutes and supernatant was discarded.
v. Tubes were kept in dessicator for overnight.
vi. On next day, the precipitate was dissolved in 4 ml GDW and mixed well.
vii. 2 ml of the above was taken and to it 0.1 ml of 80% phenol and 5 ml of
H2SO4 were added.
viii. It was mixed and cool.
ix. The absorbance was read at 624 nm spectrophotometrically against blank
solution.
Blank solution: 2 ml DDW +0.1 ml phenol reagent+ 5 ml of conc. H2SO4
Calculation: Amount of Glycogen was calculated by the following formula:
Reading of unknown
×
Concentration of standard × 1000 ×
4
Reading of standard Tissue taken 1
Where:
4/1 is volume taken.
Glycogen content was expressed as mg of glycogen/gm of tissue.
v. Superoxide Dismutase (SOD) assay:
The superoxide dismutase activity in liver was estimated by the method of
Marklund and Marklund (1974)
Principle: Pyrogallol is used to generate a reproducible O2. The estimation of
superoxide dismutase is based on inhibition of pyrogallol auto-oxidation at pH 8.0.
Material and Methods 43
Procedure:
i. To 0.1 ml tissue homogenate, 1.5 ml Tris HCl buffer was added.
ii. Then 0.5 ml EDTA was added to above.
iii. 1 ml Pyrogallol was added (during readings only)
iv. Absorbance was taken at 420 nm in spectrophotometer every minute after a
lag of 30 seconds upto 4 minutes.
Calculation: SOD activity was calculated by the following formula:
SOD = ∆ EGR / minute × 3 × 60
100× 10 µl
Where,
100= SOD enzyme activity
SOD activity was expressed as µmol/ mg protein
vi. Catalase (CAT) assay:
Catalase activity in liver was estimated by the method of Aebi (1984).
Principle: Estimation of catalase activity is based on the decomposition of H2O2.
The decomposition of H2O2 followed directly by the decrease in absorbance at 240
nm.
Procedure:
i. 2 ml of 50 mM (pH=7) phosphate buffer was added to 0.1 ml tissue
homogenate
ii. 1 ml of H2O2 (30 mM) was added to the above (during readings only).
iii. Absorbance was taken at 240 nm in spectrophotometer where readings were
taken at every 10 second interval for 30 seconds.
Calculation: CAT activity was calculated by the following formula:
CAT = ∆ EGR / minute × 3 × 60
71 × 10 µl
Where,
71= CAT enzymatic activity
CAT activity was expressed as µmol H2O2 / minute/ mg protein
Material and Methods 44
vii. Glutathione Peroxidase (GPx) assay:
Glutathione Peroxidase activity in liver was estimated by the method of
Paglia and Valentine (1967).
Principle: GPx activity was measured by the rate of GSSG formation by following
the decrease in absorbance of the reaction mixture at 340 nm as NADPH is
converted to NADP+.
Procedure:
i. 2.58 ml of 0.05 M (pH=7.0) phosphate buffer was pipette out in a quartz
cuvette.
ii. 0.01 ml tissue homogenate, 0.1 ml of 8.4 mM NADPH, 0.01 ml GR, 0.01 ml
of 1.125 M sodium azide and 0.1 ml of 0.15 M GSH was added to the above
cuvette.
iii. The reaction mixture was allowed to equilibrate at 20°C.
iv. The reaction was initiated by addition of 0.1 ml of 2.2 mM H2O2.
v. The decrease in absorbance was recorded at 340 nm between 2 to 4 minutes,
after initiation of reaction employing a cuvette with 1 cm light path in
spectrophotometer.
Calculation: GPx activity was calculated by the following formula:
GPx activity = ∆A340 × DF
6.22 × V
Where,
∆A340= A340/ min(blank)- A340/ min(sample)
6.22= mM extinction coefficient for NADPH
DF= dilution factor of sample before adding to reaction
V= sample volume in ml
GPx activity was expressed as nm of NADPH oxidized / minute/ mg protein
viii. Glutathione-S-Transferase (GST) assay:
Glutathione-S-Transferase activity in liver was estimated by the method of
Habig et al.(1974).
Material and Methods 45
Procedure:
i. 2.8 ml of 0.1 M phosphate buffer and 0.1 ml of 30 mM GSH solution was
added to 3 ml cuvette.
ii. 10-50 µl of tissue homogenate was added to the cuvette and mixed well.
iii. 0.1 ml of 30 mM CDNB solution was added to the above cuvette to initiate
the reaction.
iv. Increase in O.D. at 340 nm in a spectrophotometer was recorded every
minute for 3 minutes after a lag of 30 seconds
Calculation: GST activity was calculated by the following formula:
GST = A340(t2) – A340(t1)
(t2-t1) × (Volume (in ml) of sample added) Where,
A340(t2) = Absorbance at 340 nm at time t2 in minutes
A340(t1) = Absorbance at 340 nm at time t1 in minutes
GST activity was expressed as µ mol GSH-CDNB conjugate formed/ min/ mg
protein
Collection of blood:
Swiss albino mice were carefully dissected. The blood samples were
collected directly from the ventricle of the heart with the help of the sterilized
disposable syringe (5.0 ml) fitted with hypodermic needle. The blood samples drawn
in the syringes were immediately transferred to respective vials dipped in EDTA
solution. Sterilized plain glass centrifuge tubes were used for the separation of the
serum.
Separation of serum :
The centrifuge tubes were allowed to stand in slanting position for clotting.
After the clots have started retracting, these tubes were then centrifuged at 2,800
rpm for 30 minutes. The supernatant serum was then carefully transferred to
sterilized plain glass vials with the help of the glass dropper.
Material and Methods 46
Liver Function Tests
To assess the liver function following biochemical parameters in serum were
also done.
(i) Serum alkaline phosphatase
Serum alkaline phosphatase was measured by Kind and King's method
(1954) using commercially accessible kits (Span Diagnostics Ltd., Surat, India).
Optical density was measured at 510 nm.
Principle: Alkaline phosphatase from serum converts phenyl phosphate to inorganic
phosphate and phenol, at pH 10.0, phenol so formed reacts in alkaline medium with
4-amino antipyrine in the presence of an oxidizing agent, potassium ferricyanide,
and forms an orange-red coloured complex, which can be measured colorimetrically.
The colour intensity is proportional to the enzyme activity in the presence of an
oxidizing agent potassium ferricyanide, and forms an orange-red coloured complex,
which is measured colorimetrically.
The reaction can be represented as :
Phenyl phosphate Alkaline phosphatse Phenyl + Pi (inorganic phosphate)
pH 10.0
Phenol + 4- aminoantipyrine Potassium Phenyl + Pi (inorganic phosphate)
Ferricyanide OH_
Calculation:
Serum alkaline phosphatase activity (in KAU units)
O.D. Test - O.D. Control ×10
O.D. Std. - O.D. Blank (ii) SGOT (Serum Glutamate Oxaloacetate Transaminase) or AST
(Aspartate Transaminase)
The SGOT activity was estimated by method of Reitman and Frankel (1957)
using commercially accessible kits (Span Diagnostics Ltd., Surat, India). Optical
density was measured at 505 nm.
Principle: SGOT (AST) catalyse the following reaction:
α Ketoglutarate + L- Aspartate L – Glutamate + Oxaloacetate
Material and Methods 47
Oxaloacetate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-
DNPH) to give the corresponding hydrazone, which gives brown color in alkaline
medium and this is measured colorimetrically.
Calculation:
AST (SGOT) activity (in IU/L) = O.D. of Test - O.D. of Control
× Conc. of Std. O.D. of Std. – O.D. of Blank
(iii) SGPT (Serum Glutamate Pyruvate Transaminase) or ALT (Alanine
Transaminase)
The SGPT activity was measured by the method of Reitman and Frankel
(1957) using DNPH as colour agent by using commercially accessible kits (Span
Diagnostics Ltd., Surat, India). Optical density was measured at 505 nm.
Principle : SGPT (ALT) catalyse the following reaction :
α Ketoglutarate + L- Alanine L – Glutamate + Pyruvate
Pyruvate so formed is coupled with 2,4-dinitrophenyl hydrazine (2,4-DNPH)
to give the corresponding hydrazone, which gives brown color in alkaline medium
and this is measured colorimetrically.
Calculation:
AST (SGPT) activity(in IU/L) = O.D. of Test - O.D. of Control
× Conc. of Std. O.D. of Std. – O.D. of Blank
Haematological Study
The blood after collection in disodium EDTA vials was used for the
estimation of the haemoglobin:
Haemoglobin concentration (Hb)
The haemoglobin concentration was estimated by the standard Sahlis method
(Wintrobe et al., 1981). The method is based on the principle of making acid
haematin solution of blood in graduated haemoglobin tube and then comparing it
Material and Methods 48
with the sealed comparison tubes containing the standard acid haematin solution.
The reading was finally recorded in gm/100 ml of blood.
Statistical Analysis
Analysis of Variance (ANOVA) Test:
The statistical significance in the different enzyme activities between the
control and experimental was assessed by ANOVA. The purpose of analysis of
variance (ANOVA) is to test for significant differences between means. One way
ANOVA carried out between two groups. This test is carried by Analystsoft
Statplus 2009 software.
The significance level was obtained from the table of significance provided
for the ANOVA’s distribution. The values of P as 0.05, 0.01 and 0.001 were
considered to be almost significant, significant and highly significant, respectively.
The data generated from the above observations are expressed as the mean ± SEM.